Effect of the gln-1b mutation on nitrogen metabolite repression in Neurospora crassa.

In Neurospora crassa, synthesis of the enzymes of nitrate assimilation, nitrate reductase and nitrite reductase, was repressed by the presence of ammonium, glutamate, or glutamine. This phenomenon was a manifestation of the regulatory process termed nitrogen metabolite repression whereby alternative pathways of nitrogen acquisition are not expressed in cells enjoying nitrogen sufficiency. However, the glutamine synthetase mutant gln-1b had derepressed levels of the nitrate assimilation enzymes. The inability of glutamine to achieve nitrogen metabolite repression in this mutant militated against its potential role as the direct effector of this regulation.     

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.     

Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Nitrogen metabolism is a highly regulated process in Neurospora crassa . The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys₂/Cys₂ zinc finger DNA-binding domain. The negative-acting NMR protein interacts via specific protein–protein binding with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2–NMR protein–protein interactions. These results provide compelling evidence that the specific NIT2–NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.     

Down-regulation of eukaryotic nitrate transporter by nitrogen-dependent ubiquitinylation

In the yeast Hansenula polymorpha, the YNT1 gene encodes the high affinity nitrate transporter, which is repressed by reduced nitrogen sources such as ammonium or glutamine. Ynt1 protein is degraded in response to glutamine in the growth medium. Ynt1 disappears independently of YNT1 glutamine repression as shown in strains where YNT1 repression is abolished. Ynt1-green fluorescent protein chimera and a mutant defective in vacuolar proteinase A (deltapep4) showed that Ynt1 is degraded in the vacuole in response to glutamine. The central hydrophilic domain of Ynt1 contains PEST-like sequences whose deletion blocked Ynt1 down-regulation. Site-directed mutagenesis showed that Lys-253 and Lys-270, located in this sequence, were involved in internalization and subsequent vacuolar degradation of Ynt1. Ynt1-ubiquitin conjugates were induced by glutamine and not nitrate. We conclude that glutamine triggers Ynt1 down-regulation via ubiquitinylation of lysines in the central hydrophilic domain, and proteolysis in the vacuole.     

Phosphorylation of the yeast nitrate transporter Ynt1 is essential for delivery to the plasma membrane during nitrogen limitation

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.     

Ammonium repression of the nitrite-nitrate (nasAB) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an `out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase.     

Ammonium repression of the nitrite-nitrate ( nasAB ) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an `out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase. Received: 4 December 1996 / Accepted: 27 March 1997     

A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.     

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.     

植物铵态氮同化及其调控机制的研究进展

氮是维持植物生长发育最重要的矿质营养元素之一, 在植物整个生命进程中发挥着重要作用。在植物体内, 氮同化既是植物利用氮素的一个中心环节, 也是导致植物氮利用效率不高的因素之一。氮同化主要分为硝态氮(NO3–)和铵态氮(NH4+)同化, 其中铵态氮同化是氮同化中最为关键的一步。按照不同来源, 植物体内铵态氮同化又可分为一次同化和二次同化, 但两者都是通过谷氨酰胺/谷氨酸合成酶(GS/GOGAT)途径进行。植物铵态氮同化不仅需要大量的能量, 而且需要大量的碳源, 所以其在转录、转录后以及翻译后等各个水平上都受到严格调控。该文综述了目前关于植物铵态氮同化及其调控机制的最新研究进展。     

Xanthine dehydrogenase expression inNeurospora crassa does not require a functionalnit-2 regulatory gene

Xanthine dehydrogenase (XDH) is the initial enzyme in the purine catabolic pathway ofN. crassa. Secondary nitrogen sources such as purines are metabolized when preferred sources of reduced nitrogen (ammonium or glutamine) are unavailable. XDH synthesis is regulated by glutamine repression and uric acid induction. Thenit-2 locus is believed to encode atrans-acting positive regulator essential for the expression of genes encoding enzymes involved in secondary pathways of nitrogen acquisition, such as XDH and nitrate reductase. However, immunoblot analyses and enzyme assays reveal that XDH protein is synthesized and XDH activity is expressed innit-2 mutants. Nevertheless, XDH responds to nitrogen metabolite repression. The generality thatnit-2 is an obligate control element in nitrogen metabolite repression is questioned. Additionally, mutants defective in XDH activity, namely,xdh-1 and the molybdenum cofactor mutantsnit-1, -7, -8 and -9, are observed to grow on xanthine but not hypoxanthine.This research was supported in part by National Science Foundation Grant DMB 8516203.